THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 264, No. 26, Issue of September 15, pp. 15284-15292,1989 Printed in U. S. A.

The Chloride-activated Peroxidation of Catechol as a Mechanistic Probe of Chloroperoxidase Reactions

(Received for publication, January 26, 1989)

R. Daniel Libby$, Nicola S. Rotberg, and Jeffery T. Emerson From the Chemistry Department, Colby College, Wateruille, Maine 04901

Theresa C. White, Gabrielle M. Yen, Sharon H. Friedman, Nancy S. Sun, and Robyn Goldowski From the Chemistry Department, Barnard College, New York, New York 10027

Chloride ion (Cl-) effects on chloroperoxidase (CP0)- catalyzed peroxidation of catechol were used to probe the involvement of C1- in CPO reactions. High concen- trations of C1- inhibit catechol peroxidation by com- peting with hydrogen peroxide (ICz = 370 mM). How- ever, at lower concentrations, C1- is a linear competi- tive activator uersus catechol (KDc = 35 mM). Addition of good halogenation substrates to the peroxidatic re- action mixture converts C1- from a competitive acti- vator to a competitive inhibitor. The KZ (10 mM) for this halogenation substrate promoted C1- inhibition is equivalent to the KM (1 1 mM) for C1- in CPO-catalyzed halogenation reactions. During this inhibition, the hal- ogenation substrate is consumed and, at the point where its consumption is complete, C1- again becomes an activator. Also, at 2.0 mM hydrogen peroxide, CPOs chlorination reaction and its C1”activated peroxidatic reaction have similar apparent kc,, values.

All data are consistent with a mechanism in which C1- competes with catechol for binding to CPO Com- pound I. Catechol binding initiates the C1”independent path, in which Compound I acts as the oxidizing agent for catechol. When C1- binds to Compound I, it reacts to yield the enzymatic chlorinating intermediate which is responsible for either the oxidation of catechol in the C1”dependent path or the chlorination of substrates in the halogenation pathway. C1- activation of the per- oxidatic reaction is due to a shift from the C1”inde- pendent pathway to the C1”dependent process. The mechanism is unique in that exclusion of the substrate from its primary binding site leads to an increase in the catalytic efficiency of the reaction. This catechol- C1- system also offers further potential for probing the specificity and chemistry of the key enzymatic inter- mediates in haloperoxidase-catalyzed reactions.

Chloroperoxidase (ch1oride:hydrogen-peroxide oxidoreduc- tase, EC 1.11.1.10) is a hemoprotein with an unusually diverse range of catalytic activities. It catalyzes the reactions char- acteristic of peroxidases: hydrogen peroxide-supported oxi- dation (peroxidatic reaction (Equation 1)) and iodination (halogenation reaction (Equation 2, X- = I - ) ) of a variety of

organic substrates. Unlike most other peroxidases, CPO’ can utilize chloride and bromide in its halogenation reactions (1). This broad halide specificity is shared only by myeloperoxi- dase (2). In addition to peroxidase reactions, CPO also cata- lyzes the dismutation of hydrogen peroxide (catalatic reaction (Equation 3, R = H)), a reaction characteristic of catalases (3), and the dismutation of organic hydroperoxides and peroxy acids (Equation 3, R = alkyl or acyl groups), reactions not catalyzed by catalases or other peroxidases (4).

Hz02 + PSHz -+ PS + 2 Hz0 (1)

Hz02 + RH + X - + H* + RX + 2 Hz0 (2)

2 ROOH -+ 02 + 2 ROH (3)

Although the peroxidatic and catalatic reactions (Equations 1 and 3) catalyzed by CPO do not consume halide ions, at least some of them are accelerated by the presence of chloride (4). Thus, chloride has a broad involvement in the reactions catalyzed by CPO.

Halide ions form spectrally distinct complexes with the native forms of most peroxidases (1, 2, 5-9). Thus far, it has been difficult to determine unambiguously whether any of these complexes are part of normal peroxidase reaction path- ways since halide ions often act as both substrates and inhib- itors (7,10,11). In this paper we report the kinetic character- ization of the chloride activation of CPO-catalyzed catechol peroxidation and its use as a mechanistic probe of the specific role of chloride in chlorination reactions catalyzed by CPO.

EXPERIMENTAL PROCEDURES

General Reaction Conditions and Data Analysis-All kinetic meas- urements were made at 25 “C and at pH 2.75, the optimum pH for CPO halogenation reactions under standard reaction conditions (1). All kinetic constants were determined from weighted nonlinear least squares fits of primary kinetic data to the hyperbolic Michaelis- Menten equation using the squares of the reciprocals of the rates as weighting factors (12). Data for secondary plots were fit with a weighted linear least squares program using the squares of the recip- rocals of the dependent variables as weighting factors.

Enzyme Preparations-CPO was either provided by Professor Lowell P. Hager and Dr. Milton Axley of the Department of Bio- chemistry at the University of Illinois, Urbana, IL, or produced and purified in our laboratories as described previously (13). The enzyme

~

* This work was supported by Research Corporation Grant C-1663. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

~~ ~

$ To whom correspondence should be addressed.

~ ~

The abbreviations used are: CPO, chloroperoxidase; PSH2, gen- eral peroxidatic substrate; PS, general peroxidatic product; RH, hal- ogenation substrate; RCl, halogenated product; ROOH, hydrogen peroxide or organic hydroperoxides; EOX, enzymatic halogenating intermediate; AH,, catechol; A, catechol’s peroxidatic product; HPLC, high pressure liquid chromatography.

15284

A Probe for Chloroperoxidase Reactions 15285

had a specific activity of 1400 units/mg and an R, value of 1.4 (1). Concentrations of CPO solutions were determined from their absorb- ance at 398 nm using a molar absorption coefficient of 85,000 M”. cm”.

HPLC Separations-Chromatographic analyses were carried out using a Beckman HPLC system consisting of a Beckman l lOA pump, a Beckman System Organizer injector, a Beckman 330 UV detector, and a 25-cm Lichrosorb Rp-18 5-pm reverse phase column. A flow rate of 1 ml/min was used for all chromatographic separations.

Spectral Measurements-All UV-visible absorption measurements were determined in a Cary 118c, a Perkin-Elmer 200, a Gilford 240, or a Shimadzu UV-160 spectrophotometer using 1-cm path length quartz cuvettes.

Hydrogen Peroxide Solutions-All hydrogen peroxide solutions were prepared from 30% hydrogen peroxide, Fisher ACS Reagent. Concentrations of solutions were determined by the method of Cotton and Dunford (14).

Hypochlorous Acid Solutions-Hypochlorous acid solutions were prepared from Clorox Bleach and assayed by their ability to oxidize iodide under the conditions described by Cotton and Dunford (14).

Substrate and Product Concentration Measurements-Concentra- tions of 2-chlorodimedone solutions were determined from their ab- sorbances at 278 nm using a molar absorption coefficient of 12,200 ~ “ . c m “ (15) or at 288 nm using a molar absorption coefficient of 10,500 M“ . cm” determined under our reaction conditions. The latter wavelength is isosbestic for the peroxidatic reaction of catechol (see “Results”). Thus, 288 nm was used to measure changes of 2-chloro- dimedone concentrations in the presence of catechol and its peroxi- datic product. Rates of catechol peroxidation were determined from the increase in the absorbance at 400 nm using a molar absorption coefficient of 1300 ~ “ . c m ” . This molar absorption coefficient was determined from a plot of the absorbance versus the number of moles of hydrogen peroxide consumed in CPO-catalyzed reactions where hydrogen peroxide was the limiting reagent. Thus, it reflects the amount of hydrogen peroxide consumed by the peroxidatic reaction of catechol.

Determination of the Michaelis Constant for Chloride in CPO- catalyzed Halogenation Reactions-The apparent KM value for chlo- ride in the CPO-catalyzed halogenation reaction of 2-chlorodimedone was determined in reaction solutions containing 100 mM citrate buffer at pH 2.75, 2.0 mM H202, 83 FM 2-chlorodimedone, 0.60, 0.70, 0.88, 1.2, 1.7, 3.1, and 20 mM chloride, and 0.5 nM CPO. Rates were determined by following the decrease in absorbance at 278 nm as indicated above.

Determination of the Michaelis Constant for Catechol in the Chlo- ride-independent Peroxidatic Reaction Catalyzed by CPO-The ap- parent KM value for catechol in its chloride-independent CPO-cata- lyzed peroxidatic reaction was determined in reaction solutions con- taining 100 mM citrate buffer at pH 2.75, 2.0 mM H202, 0.02, 0.35, 0.68, 1.0, 1.35, 1.67, and 2.0 mM catechol, and 6.6 nM CPO. Rates were determined by following the increase in absorbance at 400 nm as described above.

Methionine-promoted Chloride Inhibition of Catechol Perorida- tion-Reaction conditions and data processing were identical to those used for the reactions described in Fig. 2 except that 10 mM methio- nine was substituted for 2-chlorodimedone, and the CPO concentra- tion was 3.3 nM.

Comparison of Catechol Reactions under Various Sets of Condi- tions-In each case, the progress of the catechol reaction was followed by repeatedly scanning the spectrum (500-200 nm) of the reaction mixture using the overlay mode of a Shimadzu UV-160 UV-visible spectrophotometer. The reactions and specific conditions were as follows: CPO chloride-independent peroxidatic reaction, 600 p M cat- echol, 2 mM H202, 3.3 nM CPO, and 100 mM citrate, pH 2.75; CPO chloride-activated peroxidatic reaction, 600 p~ catechol, 200 mM chloride, 2 mM H202, 3.3 nM CPO, and 100 mM citrate, pH 2.75; horseradish peroxidase reaction, 600 p~ catechol, 2 mM H20z, 3.3 pg/ ml horseradish peroxidase, and 100 mM citrate, pH 2.75; hypochlorous acid reaction, a continuous reaction was simulated by preparing 10 reaction mixtures with progressively increasing hypochlorous acid concentrations. Specific conditions used were: 600 p~ catechol, 100 mM citrate, pH 2.75, and 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, and 1.0 ptM hypochlorous acid.

Materials-All other reagents were of the highest quality available from commercial sources. Water used in these studies was glass- distilled and then further purified by passage through two activated charcoal columns, two deionizer columns, and a 0.2-p filter.

RESULTS

Substrate Effects on the Peroxidatic Reaction-At pH 2.75, the CPO-catalyzed peroxidation of catechol shows substrate inhibition by both hydrogen peroxide and catechol. Hydrogen peroxide concentrations above approximately 20 mM and catechol concentrations above approximately 5 mM produce significant inhibition. Thus, all studies where these effects are important were conducted at substrate concentrations well below these limiting values.

Chloride Effects on the Peroxidatic Reaction-At catechol concentrations below saturation, chloride activates the CPO- catalyzed peroxidation of catechol. However, the plot of the rate versus chloride concentration yields a maximum at ap- proximately 50 mM chloride. As shown in Fig. 1, at concen- trations above 100 mM, chloride acts as a weakly binding linear competitive inhibitor versus hydrogen peroxide. The Kr for chloride in this process is 370 2 25 mM.

Below 20 mM, where its inhibitory effect should be negli- gible, chloride acts as an activator of the enzymatic peroxi- dation of catechol. Repetitive UV-visible spectral scans of CPO-catalyzed catechol peroxidation reactions in the pres- ence or absence of chloride are identical. Sharp isosbestic points occur at 260.5 and 288.0 nm, and a major product peak appears a t 388 nm. Identical isosbestic points and product peaks are also obtained from catechol peroxidation reactions catalyzed by horseradish peroxidase or from reactions of catechol with hypochlorous acid under similar conditions (see “Experimental Procedures”). Also, HPLC analysis of product mixtures from CPO reactions in the presence or absence of chloride as well as the comparable horseradish peroxidase or hypochlorous acid reactions all produce single product peaks with identical retention times whether the eluting solvent is 25% aqueous methanol, 5.5 min, or 15% aqueous methanol, 6.1 min (see “Experimental Procedures” for chromatographic conditions). So, whatever its function, chloride does not change the identity of the product of CPO-catalyzed catechol reactions, and the product is the same as that formed in the horseradish peroxidase-catalyzedperoxidation of catechol and in the reaction of catechol with hypochlorous acid.

As indicated in Fig. 2, when the data for chloride activation of CPO-catalyzed catechol peroxidation are plotted with cat- echol as the variable substrate and chloride as the variable fixed substrate (16-18), chloride affects only the apparent &/kcat (slope) and not the apparent l/kcat (intercept). The activation is measurable only below approximately 20 mM catechol and must result from a decrease in the apparent KM for catechol rather than from an increase in apparent kcat. The K D ~ ? (dissociation constant: see “Appendix”) for chloride is 35 t- 8 mM and the apparent kat is 2000 & 100 s-l at 2 mM hydrogen peroxide and saturating catechol concentrations.

An analysis of the activation data treating chloride as the variable substrate and catechol as the variable fixed species (Figs. 3 and 4) shows linear variations in both the apparent &/kcat (slope) and the apparent l/kCBt (intercept) uersus the reciprocal of the catechol concentration. These plots yield an apparent KM of 27 & 8 FM for catechol and an apparent kat of 2000 ? 60 s-’ at 2 mM hydrogen peroxide and saturating chloride concentrations. An analysis done under conditions

The following constants are used. Michaelis constants: dissociation of chloride is an activator; Kppr and K p A ~ , peroxide and catechol in peroxidate reactions; Kcpr, Kcc~, and KcAH, peroxide, chloride, and catechol in chloride-dependent reactions; KHwr and KRH, peroxide and halogenation substrates in halogenation reactions; KIC,, inhibitor constant for chloride in the presence of halogenation sub- strates; KI, dissociation constant for chloride in its substrate inhibi- tion (Scheme I, Step 7). Catalytic constants: kpeet, peroxidatic reac- tion; kccat, chloride-dependent reaction; kHcet, halogenation reaction.

15286

FIG. 1. Inhibition of CPO-cata- lyzed catechol peroxidation by high chloride concentrations. Reaction conditions: 100 mM citrate, pH 2.75; 0.5, 0.8, 1.0, 2.0, and 4.0 mM H202, 10 mM catechol; 6.6 nM CPO and A, 0; M, 100; 0, 200; 0, 300; and 0, 400 mM chloride. The lines on this plot were generated using the KM and kcat obtained from a weighted least squares fit to uo/E = k,.,[HZO,]/KM + [H~OZ] (see “Experi- mental Procedures”). The apparent kcat values range from 13 f 3 to 18 f 6 ms” with a mean of 16 k 2 ms”, thus all apparent kea, values are equivalent within experimental error. A replot of the apparent KM/kcat (slope) values uer- sw chloride concentrations yields a lin- ear plot with a slope of 3.8 k 2 ps and an intercept of 1.40 f 0.05 FM.S. (Inter- cept)/(slope) = KI = 370 * 25 mM for chloride, kccat = 16 f 2 ms”, and KCper = 22 f 4 mM (see “Appendix”).

A

3 - c . w

Probe for Chloroperoxidase Reactions

I ” ” ~ ” ” ’ ” “ l “ ” ~ ’ l

1iCateChol lmM.1) FIG. 2. Chloride activation of CPO-catalyzed catechol per-

oxidation at various concentrations of chloride. Reaction con- ditions: 100 mM citrate, pH 2.75; 2.0 mM H202; 0.10, 0.20, 0.30, 0.40, 0.50, 0.80, and 1.10 mM catechol; 2.7 nM CPO and 0, 2.0; 0, 5.0; 0, 8.0; M, 11.0; A, 14.0; A, 17.0; 0, 20 mM chloride. The lines on this plot were generated from apparent KM and kcat values obtained from a weighted least squares fit to uo/E = kC,.,[catechol]/KM + [catechol] (see “Experimental Procedures”). The apparent keet values vary from 2.0 2 0.1 to 2.2 * 0.2 ms” with a mean of 2.0 k 0.1 ms”; thus all apparent kcat values are equivalent within experimental error. A replot of the apparent KM/kCat (slope) values uersus l/chloride concentration yields a linear plot with a slope of 460 f 30 p&.s and an intercept = 13 f 2 nM.s. (Slope)/(intercept) = KDC, = 35 f 8 mM for chloride as an activator, Kcper = 14 -C 5 mM, and KCAH = 220 f 80 p M (see “Appendix”).

similar to those of Fig. 2 but in the absence of chloride (see “Experimental Procedures”) yields an apparent KIM of 2.4 f 0.3 mM for catechol at 2.0 mM hydrogen peroxide. Thus, chloride activates by lowering the apparent KM for catechol by a factor of 100 without increasing kcat for the reaction. The kinetic relationship between chloride and catechol can be

llCa1echol (mM-1)

J

FIG. 3. Slope replot of the chloride-activated peroxidation of catechol by CPO at various catechol concentration. This is a replot of apparent KM/kat values from a weighted least squares fit to uo/E = &,,,[ch1oride]/KM + [chloride] (see “Experimental Proce- dures”). Reaction Conditions: 100 mM citrate, pH 2.75; 2.0 mM H202; 2.0, 5.0, 8.0, 11.0, 14.0, and 20.0 mM chloride; 0.1, 0.2, 0.3, 0.4, 0.5, 0.8, and 1.10 mM catechol; and 2.7 nM CPO. The slope of the replot is 470 f 30 pM2.s, the intercept is -0.11 f 0.05 mM.S, and KCAH = 210 f 90 pM (see “Appendix”).

described by the following empirical equation.

This is the kinetic pattern expected for ordered binding of two substrates where the rate of release of the first substrate (chloride in this case) is much greater than the rate limiting step (19). However, the catechol-chloride reaction mechanism must be more complicated. Catechol reacts in the absence of chloride and since chloride has no effect on the reaction at high catechol concentrations, chloride and catechol must com- pete for binding to the same enzymatic intermediate at some point in the reaction pathway. Thus, chloride might be termed

A Probe for Chloropt

a Competitive actiuator of CPO-catalyzed catechol peroxida- tion.

To clarify the mechanism of activation and its possible relationship to chlorination reactions catalyzed by CPO, the chloride effect on catechol peroxidation was investigated in the presence of halogenation substrates. 2-Chlorodimedone, a good substrate for CPO halogenation reactions, greatly mod- ifies the effect of chloride on the catechol reaction (see Fig. 5 ) . Saturating concentrations of 2-chlorodimedone convert chloride from a competitive activator to a linear competitive inhibitor versus catechol. The apparent KIcI (see “Appendix”) for chloride in these reactions is 10 2 2 mM. Identical results

0 7

t 1

llcatechol (mM-1)

FIG. 4. Intercept replot of the chloride-activated peroxida- tion of catechol by CPO at various catechol concentration. This is a replot of apparent l/k,,, values from the primary plots described in Fig. 3. The slope of the replot is 14 f 4 nM.s and intercept = 510 f 14 ps. (Slope)/(intercept) = apparent K, = 27 f 8 p~ for catechol in the chloride-dependent peroxidatic reaction (ap- parent KCAH), the apparent kat for the reaction is 2000 f 60 s-’, KCAH = 220 f 90 pM, and Kcper = 14 f 2 mM. Also, (slope, Fig. 3)/(slope, Fig. 4) = KDCI = 34 f 12 mM (see “Appendix”).

FIG. 5. Halogenation substrate promoted chloride inhibition of cat- echol peroxidation. Reaction condi- tions: 100 mM citrate, pH 2.75; 2.0 mM HzOz, 82 PM 2-chlorodimedone; 0.35, 0.70, 1.0, 1.35, and 2.0 mM catechol; 3.3 nM CPO and 0, 0; 0, 2.0; 0, 3.5; ., 5.0; A, 10.0; A, 20.0 mM chloride. The lines on this plot were generated using K, and kcat values obtained from a weighted least squares fit to UO/E = k,.,[catechol]/K~ + [catechol] (see “Experimental Proce- dures”). The apparent kat values vary from 2.2 f 0.1 to 2.8 f 0.3 ms” with a mean of 2.5 f 0.3 ms”, thus all apparent kcat values are equivalent within experi- mental error. A replot of the apparent K,+,/k,., (slope) values uersus chloride concentrations is linear with a slope of 43 & 6 ps and an intercept of 420 f 40 nM.s. (Intercept)/(slope) = K1cl = 10 f 2 mM for chloride in the presence of 2- chlorodimedone, KpPr 5 11 -C 3 mM, and KpAH 5 7 & 2 mM (see “Appendix”).

P - > . w

mxiduse Reactions 15287

(apparent KIc, = 10 f 2 mM) were also obtained using methi- onine as the halogenation substrate (see “Experimental Pro- cedures”).

Fig. 6 shows the progress curve of a representative reaction containing 2-chlorodimedone. During the initial phase of the reaction, both catechol and 2-chlorodimedone are being con- sumed and the rate of catechol peroxidation is inhibited. However, at the point where 2-chlorodimedone is completely consumed, the catechol peroxidation rate increases sharply. After this point, the peroxidation rate is that expected for the chloride-activated reaction in the absence of halogenation substrates. Also, in the early phase of this reaction, the sum of the rates of the catechol peroxidation (26 f 1 mM/min) and the halogenation reaction (87 f 4 mM/min) is equal, within experimental error, to the rate of catechol peroxidation in a similar reaction mixture containing no 2-chlorodimedone (109 f 5 mM/min). Thus, 2-chlorodimedone quantitatively replaces catechol as a substrate for part of the enzymatic reaction.

Table I summarizes all values of kinetic constants deter- mined from various plots of our data. The agreement of values obtained from different data sets or different plots of the same data set give an indication of the self-consistency of our results.

Comparison of Peroxidatic and Halogenation Reactions- The effects of 2-chlorodimedone and methionine seem to indicate that the CPO halogenation pathway is involved in the chloride activation of catechol peroxidation. To investi- gate the kinetic competence of the halogenation process as an activation path, a comparison was made between rates of 2- chlorodimedone halogenation and catechol peroxidation at high chloride concentrations. The chloride concentrations used were in the inhibitory range for both sets of reactions and well above the apparent KM (apparent KHcl = 11 mM) for chloride in the halogenation process (see “Experimental Pro- cedures” for reaction conditions) and K D ~ , (35 mM) for chlo- ride in the peroxidation of catechol (see Table I). Table I1 shows that at these high concentrations of chloride the ap-

5 0

4.5

4.0

3.5

3.0

2 5

2 0

1.5

1 .o

0 5

0.0 0. 0 0 . 5 1.0 1.5 2.0 2.5 3 0

YCateehol (ms)

15288 A Probe for Chloroperonidase Reactions

parent Kc, , for the peroxidation of catechol is essentially identical to that for the halogenation of 2-chlorodimedone under similar conditions. The rates of both sets of reactions in Table I1 are independent of the concentrations of the organic substrate (catechol or 2-chlorodimedone). Thus, the reactions are at or very close to saturating concentrations of these substrates.

DISCUSSION

All of our data are consistent with the proposed general mechanism for CPO-catalyzed reactions outlined in Scheme

100

E v E W z x s I: a

0,

x

50

I

0 . 5 1 . 0 1 . 5

MINUTES

FIG. 6. 2-Chlorodimedone and chloride effects on catechol peroxidation. Reaction conditions: 100 mM citrate, pH 2.75; 2.0 mM H202; 0.35 mM catechol; 82 PM 2-chlarodimedone; 10 mM chloride; and 1.33 nM chloroperoxidase. Traces show changes in the concen- tration of W, 2-chlorodimedone, and 0, peroxidatic product from catechol. The dashed line indicates the point at which the 2-chloro- dimedane concentration reaches zero.

I. Step 7 accounts for inhibition by high concentrations of chloride. The chloride-independent path is represented by Steps 1-3, the chloride-dependent pathway for the peroxidatic reaction is represented by Steps 1 and 4-6 and the chlorina- tion reaction is represented by Steps 1, 4, 8, and 9. Steps 3 and 6 may include several enzymatic intermediates (e.g. Com- pound I1 and related species); however, our data yield no information about these steps, so we have grouped them together for simplicity. Thus, Steps 1 and 7 are common to all CPO reactions, and Step 4 is common to the chloride- dependent peroxidatic and halogenation reaction pathways. We propose that chloride activation of catechol peroxidation is caused by a shift in mechanism from the chloride-inde- pendent to the chloride-dependent pathway. We will discuss our results in terms of the mechanism proposed in Scheme I (see "Appendix" for kinetic equations derived from Scheme I).

Although chloride acts as an inhibitor as well as a substrate or activator in CPO-catalyzed reactions, the negative effects can be studied separately from the positive effects. The inhi- bition occurs only at very high chloride concentrations (KI = 370 mM, Fig. l), where the activation path = 35 mM, Table I) is saturated. The activation effect can be studied a t moderate chloride concentrations where the extent of inhibi- tion should be small. Also, the sites at which chloride exerts these two effects are different. Thus, we will consider each effect separately.

At concentrations above approximately 100 mM, chloride acts as a competitive inhibitor versus hydrogen peroxide in the CPO-catalyzed peroxidation of catechol (Fig. 1). For all peroxidases, it is generally accepted that hydrogen peroxide binds to the heme group of the native enzyme. It then reacts with the heme iron to produce the enzymatic intermediate designated Compound I (20). Thus, as a competitive inhibi- tor, chloride probably binds a t or near the heme of the na- tive CPO inhibiting the formation of Compound I (Scheme I, Step 7).

Lambier and Dunford (11) have proposed that chloride is a competitive inhibitor of the formation of CPO Compound I. They used indirect evidence, chloride inhibition of cyanide binding to the heme group of native CPO, to support their argument for competition between chloride and hydrogen peroxide. Their K, for chloride (approximately 375 mM at pH 2.75, interpolated from their graph of KI uersus pH) is very similar to our value. Our direct kinetic evidence of chloride competition with hydrogen peroxide in its inhibition of the peroxidation of catechol at high chloride concentrations proves conclusively that weak binding of chloride to native

TABLE 1 Summary of kinetic constants

See "Appendix" for methods used to determine these constants. Constant Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Results

A Probe for Chloroperoridase Reactions 15289

CPO (3, 9-11) is not kinetically productive. Thus, although the pH dependence of spectrally detectable chloride binding to native CPO parallels the pH dependence of CPO halogen- ation activity (3), the chloride complexes formed with native CPO must be the chloride-inhibited form of CPO indicated in Step 7 of Scheme I.

The chloride activation effect, which is observed below 20 mM chloride is significant only at subsaturating concentra- tions of catechol. The lack of a chloride effect on the apparent kc,, when catechol is saturating (see Fig. a) , suggests that chloride competes with catechol for binding to a single form of the enzyme and that chloride binding is completely blocked by saturating catechol concentrations. However, the depend- ence of the apparent kc,, on catechol concentration when chloride is saturating (see Fig. 4) indicates that catechol binding cannot be completely suppressed by chloride. The simplest explanation for these data is that catechol must bind productively to two different forms of CPO in the reaction pathway. One form binds both chloride and catechol and the other binds only catechol.

In the generally accepted mechanism for horseradish per- oxidase-catalyzed peroxidatic reactions, the organic substrate (catechol) binds to Compound I (20, 21). In CPO-catalyzed chlorination reactions, chloride reacts with Compound I pro-

TABLE I1 Apparent catalytic rate constants of CPO-catalyzed peroxidatic and

halogenation reactions at high chloride concentrations General reaction conditions: 100 mM citrate, pH 2.75; 2.0 mM

K O , . Apparent k,.P

Peroxidation* Halogenation‘ [Chloride]

?7lM ms” 100 1.60 +. 0.07 200

1.55 ? 0.05 1.23 f 0.03

300 1.25 & 0.03

1.03 2 0.02 1.15 & 0.05 a The apparent kcat values shown are averages of values determined

* Peroxidation: 1 mM catechol, 1.7, 3.3, and 6.6 nM CPO. at the three enzyme concentrations listed in Footnotes b and c.

CPO. Halogenation: 82 p M 2-chlorodimedone, 0.53, 1.1, and 1.6 nM

ducing EOX (the enzymatic electrophilic halogenating inter- mediate; see Scheme I, Step 4). EOX then transfers chlorine to the organic substrate as illustrated in Scheme I, Step 9 (11, 22-24). Using transient state kinetics, Dunford et al. (24) showed that, in the absence of other substrates, chloride catalyzes the decomposition of CPO Compound I. They inter- preted their data in terms of a halogenation mechanism in which chloride binds to Compound I rather than to the native form of the enzyme.

We suggest that in the CPO reaction, chloride and catechol compete for binding to Compound I (Scheme I, Steps 2 and 4). Chloride binding allows the formation of an enzymatic halogenating intermediate, EOX, which provides the second binding site for catechol (Scheme I, Step 5 ) . When chloride concentrations are increased the reaction path shifts toward EOX (Scheme I, Step 4). Since EOX has a lower apparent KM (apparent KCAH = 27 pM) for catechol than does CPO Compound I (apparent K ~ A H = 2.4 mM), the shift of path toward EOX decreases the apparent KM for catechol produc- ing the observed activation. On the other hand, when the catechol concentration is raised high enough to saturate its Compound I binding site, chloride can no longer bind to its site on Compound I and the activation vanishes. Thus, chlo- ride is a KM or competitive activator of the CPO-catalyzed peroxidation of catechol. This mechanism has the interesting characteristic that when catechol is excluded from its primary binding site, the enzyme’s catalytic efficiency (kcat/&) for catechol peroxidation increases (see Fig. 2).

Involvement of EOX in the peroxidatic reaction is con- firmed by the effects of methionine and 2-chlorodimedone (Fig. 5 and “Results”). These halogenation substrates (Scheme I, Steps 8 and 9) rapidly and efficiently consume EOX in CPO reactions (22). Consequently, such substrates should exclude catechol from the activation path (Scheme I, Steps 5 and 6) and convert chloride into a competitive inhib- itor versus catechol. Fig. 6 shows that 2-chlorodimedone is consumed while it promotes chloride inhibition of the perox- idatic reaction. The linearity of the peroxidatic reaction pro- gress curve as 2-chlorodimedone is consumed (see Fig. 6, up to the vertical dashed line) indicates that, even at its very

CI- CI

/

GQ”u

SCHEME I. Proposed reaction mechanism for chloroperoxidase- catalyzed reactions.

A + H20

/ ‘ CI

L L

0-

15290 A Probe for Chloroperoxidase Reactions

lowest measurable concentrations, 2-chlorodimedone effec- tively blocks the chloride-dependent pathway. If catechol had begun to compete successfully with 2-chlorodimedone as the reaction progressed, the rate of peroxidation would have in- creased with time and the progress curve for peroxidatic product formation would have been concave upward. The sharp increase in the rate of peroxidatic product formation when the 2-chlorodimedone concentration reaches zero, in- dicates that only in the absence of the halogenation substrate can the chloride-dependent pathway produce peroxidatic product from catechol.

Therefore, the pathway which, in the absence of the halo- genation substrates, leads to activation of the catechol reac- tion (Scheme I, Steps 4-6), becomes a deadend pathway for catechol in their presence (Scheme I, Steps 4, 8, and 9). This deadend pathway is the normal CPO halogenation process. Thus, in the presence of 2-chlorodimedone, the apparent KIcl for chloride inhibition should be equal to the apparent KM for chloride (apparent in the CPO-catalyzed chlorination reaction (see derivation under “Appendix”). The values found (apparent KICI = 10 * 2 mM and apparent KHcl = 11 k 1 mM) are equivalent. This is conclusive evidence that chloride uti- lizes the same binding site on CPO Compound I in both the halogenation and the chloride-dependent peroxidatic path- ways.

The proposed mechanism also predicts that halogenation substrates can be used to dissect the peroxidatic reaction into chloride-dependent and -independent parts. Halogenation substrate consumption (Scheme I, Steps 8 and 9) would monitor the chloride-dependent path, while catechol con- sumption (Scheme I, Steps 2 and 3) monitors the chloride- independent path. The quantitative relationship between per- oxidatic and halogenation rates in the early phase of the reaction in Fig. 6 (see “Results”) confirms this prediction.

The data in Figs. 3 and 4 show that at saturating chloride concentrations catechol peroxidation proceeds through a se- quential bireactant mechanism (16-19). Under these condi- tions, the Compound I binding site for chloride (Scheme I, Step 4) is saturated and catechol peroxidation is proceeding exclusively through the chloride-dependent pathway (Scheme I, Steps 4-6). This is a sequential pathway with ordered chloride and catechol binding. Finally, the equivalence of the rates of chlorination and peroxidation at very high chloride concentrations (see Table 11) indicates that the halogenation path is kinetically competent to account for chloride activa- tion of the peroxidatic reaction.

The data reported in this paper provide a relatively clear understanding of the kinetic mechanism through which chlo- ride activates the CPO-catalyzed peroxidation of catechol. It is the first documented case of an activator which actually competes with the substrate of the reaction which is activated. Chloride blocks catechol binding to CPO Compound I, but in doing so it produces a new enzymatic intermediate, EOX, which has a higher affinity for catechol than does Compound I. The result is a unique case of competitive or K, actiuation.

In addition, since the activation mechanism is intertwined extensively with the mechanism of CPO-catalyzed chlorina- tion reactions, our results allow us to draw some general conclusions concerning all chloride-dependent reactions cat- alyzed by CPO. 1) CPO Compound I provides the catalytic binding site for chloride in all chloride-dependent reactions. 2) Chloride binding to native CPO definitely inhibits CPO reactions and is not responsible for positive chloride effects in any CPO reactions including chlorination reactions. 3) The EOX intermediate is responsible for oxidation of catechol in the chloride-dependent path and probably also participates in

chloride activation of CPO’s catalase reaction (3). So, just as halogenation substrates allowed us to dissect the peroxidatic process, peroxidatic substrates can be valuable probes of CPO halogenation and catalatic processes (1, 3, 4).

Since peroxidatic substrates are oxidized by CPO Com- pound I, these substrates could be used to explore similarities and differences between the reactivities of CPO Compound I and those of other peroxidases. Does CPO Compound I oxidize substrates by a one- or a two-electron process or is the mechanism variable as has been reported for thyroid peroxi- dase (26)?

Since EOX is responsible for oxidation of the substrate in chloride-dependent processes, peroxidatic substrates could be used to investigate the chemical behavior of EOX. It is re- markable that products from Compound I and EOX oxidation of catechol are identical. One might expect EOX, which is an electrophilic halogenating species, to oxidize organic sub- strates by electrophilic halogenation followed by dehydrohal- ogenation, two ionic reactions. This would suggest that cate- chol reacts with Compound I through an ionic two-electron pathway. However, it is possible that both radical and ionic processes produce the same product. It should be noted that horseradish peroxidase produces the same catechol product (see “Results”) and that in CPO reactions with some other peroxidatic substrates, spectra of product mixtures produced in the presence of chloride are different from those obtained in its ab~ence .~ Thus, the chemical course of CPO peroxidatic reactions may be sensitive to the structure and reactivity of the substrate.

We believe that the reaction we have characterized in this report offers the potential to answer many questions about CPO reactions. Can EOX act as a one- or two-electron oxi- dant? To what extent is free hypohalous acid involved in CPO halogenation or halide dependent reactions? Thus, this chloride-activated system will be a valuable tool for investi- gating the mechanisms of reactions catalyzed by CPO and other haloperoxidases.

APPENDIX

Derivation of Kinetic Equations f rom Mechanism in Scheme I

The kinetic mechanism illustrated in Scheme I is a combi- nation of three submechanisms: the chloride-independent per- oxidatic reaction (Steps 1-3), the chloride-dependent peroxi- datic reaction (Steps 1 and 4-6) and the halogenation reaction (Steps 1, 4, 8, and 9). All of these processes are potentially subject to inhibition by chloride through Step 7. To analyze the complete scheme it is useful to derive the kinetic equation in terms of the kinetic constants that describe each of the submechanisms. In our derivations, the rate constants are identified by subscripts which correspond to the number of their step in Scheme I. Positive subscripts indicate constants for the forward direction of the step, and negative subscripts indicate constants for the reverse direction.

Chloride-independent Pathway (Steps 1-3)”This mecha- nism is a ping-pong bireactant process which can be described by initial velocity Equation 5:

where kPcat = kS, KPAH = (k+ + kd/k2 and Kpper = k3/k1. At 2 mM H202 the apparent kcat = kPcat[H2021/(K+, + [HzOzl) and

R. Goldowski, N. S. Sun, J. T. Emerson, and R. D. Libby, unpublished result.

A Probe for Chloroperoxidase Reactions 15291

the apparent KM for catechol, apparent KPAH, is KPAH[H~O~]/ (K~per + [HzOzl).

Chloride-dependent Pathway (Steps 1 and 4-@-This re- action is a ping-pong trireactant process which can be de- scribed by initial velocity Equation 6:

" = kceat[H~0zlIC11IAHzl E (KDCIKCAH[HZOP] + KCAH[HZO~][CI] 4- Kcc~[HzOzl[AH~l (6)

+ KcpJCIl[AH2I + [H~OZI[C~I[AH~I)

where kccat = ks, KDc, = k+/k4, KCAH = (12-5 + ks)/k5, Kcper = k d k l , and KCCI = kdk4.

Halogenation Reaction (Steps I , 4, 8, and 9)"This reaction is also a ping-pong trireactant process which can be described by initial velocity Equation 7:

uo

E (KDcIKRH[HYO~] + KRH[HzOZ] [Cl] + K~c~[Hzoz] [RH] (7) " ~HC~L[H~OZ][CI~[RHI -

+ KH,.[C~][RH] + [H2O~I[C1I[RHl)

where h a t = ks, KDCI = W k 4 , KRH = (k-8 + kd/k8, K H ~ ~ ~ = k 9 / k , and KHCr = k9/k4. Most studies are carried out at 2 mM H202 and at saturating levels of RH where KR~/[RH] = 0. Under these conditions, the apparent kc,, = ~ H ~ ~ ~ [ H ~ O ~ ] / ( K H ~ ~ ~ + [H202]) and the apparent KM for chloride, apparent KHc1, is KHCI[HZOP]/(KH~.I. [H2021).

Substrate Inhibition by Chloride-For all processes the in- hibition produced by chloride binding to the native enzyme (Step 7) is described by KI = k-?/k7.

Combined Chloride-independent and -dependent Reactions (Steps I-7)"Under the reaction conditions described in Figs. 1-4 (Steps 1-7) operate and yield initial velocity Equation 8:

" -~

. . .. .

+ KDCIKCAHKP~~~[C~I + Kcc&~per[C1I[AHzl KPAHKI

Kcper[C1lz KI

+- + (Kc,, + [HzO,I)[Cll) .[AH21 + K C A H [ H ~ ~ Z I [ C ~ ] 1 This equation can be reduced to a form similar to Equation 4 (see Equation 11 below). The coefficient of the numerator consists of three terms and that of [AH2] in the denominator consists of seven terms. If the first two terms of the sum in the numerator and the first five terms of the [AH,] coefficient are considered small, then Equation 8 becomes Equation 9.

As is indicated below, when our data are fit to Equation 9, reasonable and self-consistent kinetic constants are obtained (see Table I). Also, values of kinetic constants obtained from our fits to Equation 9 indicate that the terms neglected in obtaining the equation are in fact at least a factor of 10 smaller than those which were retained.

Equations Relating to Fig. 1 "Under the conditions of Fig. 1, [AH,] and [Cl] are relatively high so that &AH/[AHP] = 0

and KDC~/[CI] = 0, and Equation 9 reduces to Equation 10.

VO

E - = kccat[HnOzl

K c p e r ( g + 1) + [Hz021 (10)

Thus for the data in Fig. 1, kcat = kccat = 16 f 2 ms" and the apparent KM for H202 is Kcpei ([Cl]/K, + 1). The replot of &/kc,, uersus [Cl] yields a slope of Kcp,,/kcCatKr = 3.8 f 2 ps" and an intercept of Kcpe,/kccat = 1.4 f 0.05 p ~ . s. The K1 is obtained from intercept/slope = 370 f 25 mM. Using the values for kccat and the intercept, Kc,,, is found to be 22 f 4 mM .

Equations Relating to Figs. 2-4-Under the reaction con- ditions of Figs. 2-4, [Cl] is low relative to KI (360 mM) so that [CI]/KI = 0 and [HzOz] is constant; thus Equation 9 reduces to Equation 11 which has the form of Equation 4.

For the primary plots of the data used for Fig. 2, the

and the

Thus, for the lines in Fig. 2, the slopes = (&AH/kCeat) (KDCI/ [Cl] + 1) and the intercepts = (Kcper + [H2O2])/[H2O2]kcCat. For the replot ofthe slopes versus l/[Cl], the slope = KcA&&l/ kccat = 460 f 30 ,.LM'.s, the intercept = = 13 f 2 nM . s, and (slope)/(intercept) = KDc~ = 35 f 8 mM. Also, using kceat = 16 & 2 ms" (from Fig. l), the following constants are obtainable from this plot: Kcper = 14 f 5 mM and KCAH = 220 f 80 pM.

For Fig. 3, the slope of the primary plot = KDcIKcAH/ kCat[AH2] and the slope of Fig. 3 = KDC&AH/kCcat = 470 f 30 PM'. s. Using the kccat obtained from Fig. 1 and the KDc, obtained from Fig. 2, we determined KCAH = 210 f 90 p ~ .

For Fig. 4, the intercept of the primary plot = (KCAH/ kcCat[AH~1) + (Kcper + [ H ~ O ~ I ) / ~ C ~ ~ ~ [ H Z O S I . Thus for the replot depicted in Fig. 4, the slope = KCAH/kCcat = 14 f 4 nM.s, the intercept = (Kcper + [Hz0z])/k~,.t[H202] = 510 f 14 ps, and the apparent KM for catechol, apparent KCAH, is (slope)/ (intercept) = K C ~ ~ [ H ~ O ~ ] / ( K C , , , + [H202]) = 27 f 8 p ~ . Also, using kceat = 16 f 2 ms" (from Fig. l), values of KCAH = 220 f 90 p~ and Kcper = 14 f 2 mM are obtained. Finally, (slope, Fig. 3)/(slope, Fig. 4) = KDCI = 34 f 12 mM.

Equations Relating to Fig. 5-In catechol reactions contain- ing halogenation substrates, Steps 1-4 and 7-9 operate and chloride acts as an inhibitor even at low concentrations (Fig. 5 ) . The initial velocities of these reactions are described by Equation 12.

(KDCIKRH + KHCI[RHI) kPcat [HZOZ][AHZI UU KPAH

[ (KDCIKRH[HZOZI) (K~per + [HZOZI)K,,,[AHZI[RH] KPAH

15292 A Probe for Chloroperoxidase Reactions

Since our studies with halogenation substrates utilized satu- rating concentrations of these substrates and were carried out at constant [H202], Equation 12 can be simplified to Equation 13.

The slopes of the lines in Fig. 5 are (KPAH/kPcat) [((KH,, + [Hz02])[Cl]/[H202]K~~,) + 11 and the intercepts = + [H2Oz])/[H202]k~~~~) = 400 f 50 ps . Thus for the replot of the slopes uersus [Cl], the slope = K P A H ( K H ~ ~ ~ + [H2Oz])/kpCat. &~,[H202] = 43 f 6 p s , the intercept = KpAH/kpeat = 420 f 40 nM .s and the apparent KIc1, the inhibitor constant for chloride, is (Intercept)/(slope) = & I C ~ [ H ~ O ~ ] / ( K ~ ~ ~ ~ + [HzOz]) = 10 1- 2 mM which is identical to the apparent KM for chloride (apparent KHcl = 11 1- 2 mM), in the halogenation reaction (see Equation 7). Assuming that kpcat is no larger than kceat (16 -C 2 ms”), KpPr 5 11 f 3 mM, and K ~ A H 5 7 f 2 mM.

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